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Lymphocyte colonization by gammaherpesviruses (HVs) is an important target for cancer

Lymphocyte colonization by gammaherpesviruses (HVs) is an important target for cancer prevention. analyses are limited in their sampling and capacity to establish cause and effect. Therefore, resolving the discrepancy is not straightforward. Related HVs provide another source of information. Those that infect experimentally tractable mammals are particularly useful for establishing cause and effect in a realistic context. Murid herpesvirus 4 (MuHV-4) is a well-characterized example. Despite immortalizing only fetal B cells (5), it colonizes adult lymphoid GCs (6) to establish a persistent infection of memory B cells (7,C9). The Kaposi’s sarcoma-associated herpesvirus (KSHV) also colonizes B cells (10) and fails to transform them but remain strikingly similar in host colonization. MuHV-4 therefore provides an opportunity to understand functionally in INK 128 inbred laboratory mice how many HVs may interact with B cells (11,C13). There is no guarantee that every HV acts in the same way, but with MuHV-4 we can establish a relatively complete functional framework onto which the more fragmented information about human infections can be mapped. MuHV-4 drives B cell activation and proliferation greatly in excess of antigen-specific responses (14, 15). However, both depend on CD4+ T cells (16), CD40 ligand (17), and CD40 (18), implying a similar need for T cell-derived survival signals. Antigen-specific responses also require T cell-independent survival signals, of which those delivered by B cell-activating factor (BAFF) through its main receptor (BAFF-R) have central importance (19, 20). The BAFF-R-deficient phenotype was defined first in AsWyn/J mice (21), in which C-terminal receptor disruption creates a dominant negative mutant (22): transitional B cells developing in the bone marrow fail to survive or undergo T1 to T2 maturation. BAFF-R is also required for follicular B cell survival. Thus, competition for limiting amounts of BAFF regulates circulating B cell numbers. INK 128 B1 B cells are preserved without BAFF-R, but B2 numbers are severely reduced and marginal-zone B cells are essentially absent (23). IgM responses are still made, but GCs form only transiently and IgG responses are weak (24, 25). Targeted BAFF-R (26) and BAFF knockouts show similar phenotypes (20). BAFF-R signaling works in part through the induction of antiapoptotic family members (27). HVs encode homologs and inhibit mitochondrial apoptosis pathways (28), INK 128 so infected B cells might be expected to show independence of BAFF-R-mediated homeostatic control; conversely, extensive reliance on normal B cell physiology (29) would keep virus-driven lymphoproliferation BAFF-R dependent. Therefore, to understand better how HV host colonization works, we determined the extent to which it depends on BAFF-R. MATERIALS AND METHODS Mice. C57BL/6J (Harlan U.K.) and BAFF-R?/? mice (26) (kindly provided by Andrew Sage and Lauren Baker, Division of Cardiovascular Medicine, Cambridge University Medical School) were maintained at the Cambridge University Department of Pathology animal unit and INK 128 infected with MuHV-4 when 6 to 12 weeks old, either intranasally (i.n.) in 30 l of Dulbecco’s modified Eagle’s medium (DMEM) under isoflurane anesthesia Rabbit polyclonal to AKIRIN2 (104 PFU) or intraperitoneally (i.p.) in 100 1 of DMEM (105 PFU). All animal experiments were approved by the Cambridge University Ethical Review Board and by the 1986 Animal Scientific Procedures Act (project license 80/2538). Cells and viruses. BHK-21 cells (American Type Culture Collection CCL-10) and 3T3-ORF50 cells (30) were grown in Dulbecco’s modified Eagle’s medium, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal calf serum (PAA Laboratories). Wild-type (WT) and EF1-eGFP MuHV-4 (31) were grown on BHK-21 cells, and their titers were determined. ORF50-deficient MuHV-4 was grown on and its titer determined on 3T3-ORF50 cells (30). Virions were harvested from infected cell supernatants by ultracentrifugation (35,000 test unless stated otherwise. Viral genome quantitation. MuHV-4 genomic coordinates 4166 to 4252 were amplified by PCR from 50 to 80 ng DNA of organ homogenates (Rotor-Gene 3000; Corbett Research). PCR products were quantitated by hybridization with a TaqMan probe (genomic coordinates 4218 to 4189) and converted to genome copies by comparison with a standard curve of cloned plasmid template amplified in parallel. Cellular DNA was quantitated in the same reaction by amplifying part of the adenosine phosphoribosyl transferase (APRT) gene, again with TaqMan probe hybridization and template dilutions amplified in parallel. Viral DNA loads were then normalized by the cellular genome copy number of each sample (32). Immunohistochemistry and hybridization. Spleens were fixed in phosphate-buffered saline (PBS)C4% formaldehyde (24 h; 4C), dehydrated in 70% ethanol, and embedded in paraffin. Seven-micrometer sections were dewaxed in xylene and hydrated in graded ethanol solutions. Endogenous peroxidase activity was quenched in PBSC3% H2O2 (10 min; 23C). Sections were then blocked with an avidin/biotin blocking kit (Vector.